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目的:降低鼠源性抗血小板单克隆抗体SZ-2的免疫原性,为其进一步研究、应用奠定基础。方法:用Trizol试剂从SZ-2杂交瘤细胞抽提RNA,应用RT-PCR技术,扩增出SZ-2重链、轻链可变区基因,克隆人载体pUCm-T,测序分析。应用基因重组技术,将SZ-2轻、重链可变区基因与人免疫球蛋白γ1重链CH1和κ轻链恒区基因进行拼接,构建人-鼠嵌合Fab片段基 因表达质粒pSW1-2 Fab/Hu,并导入大肠杆菌XL1-blue诱导表达。以ELISA、Western blot和瑞斯托霉素诱导血小板聚集抑制试验,对表达产物进行检测、验证。结果:克隆的基因序列符合小鼠轻、重链可变区基因的特征;表达质粒pSW1-2Fab/Hu拼接正确;在大肠肝菌中的表达量约为180μg/L;表达产物具有与血小板GPIb结合的特性并可抑制瑞斯托霉素诱导血小板聚集。结论:成功地克隆了SZ-2可变区基因;并表达了可溶性人-鼠嵌合Fab基因工程抗体。
Objective: To reduce the immunogenicity of murine anti-platelet monoclonal antibody SZ-2 and lay the foundation for its further study and application. Methods: Trizol reagent was used to extract RNA from SZ-2 hybridoma cells. RT-PCR was used to amplify the heavy chain and light chain variable region of SZ-2. The human pUCm-T vector was cloned and sequenced. The recombinant plasmid pSW1-2 was constructed by splicing the light and heavy chain variable region genes of SZ-2 with the constant regions of CH1 and κ light chain of human immunoglobulin γ1 heavy chain. Fab / Hu and introduced into E. coli XL1-blue to induce expression. ELISA, Western blot and ristocetin induced platelet aggregation inhibition test, the expression product was tested and verified. Results: The cloned gene was in line with the characteristics of mouse light and heavy chain variable region genes. The expression plasmid pSW1-2Fab / Hu was spliced correctly. The expression level in E. coli was about 180μg / L. Binding properties and can inhibit ristocetin-induced platelet aggregation. Conclusion: The SZ-2 variable region gene was successfully cloned and soluble human-mouse chimeric Fab gene engineering antibody was expressed.